Scleroderma is a disease of the connective tissue characterized by fibrosis of the skin and internal organs, leading to organ failure and death (Black et al., 1998; Clements and Furst, 1996). Scleroderma has a spectrum of manifestations and a variety of therapeutic implications. It comprises localized scleroderma, systemic sclerosis, scleroderma-like disorders, and Sine scleroderma (Smith, 2000). Whilst localized scleroderma is a rare dermatologic disease associated with fibrosis and manifestations limited to skin, systemic sclerosis is a multisystem disease with variable risk for internal organ involvement and variation in the extent of skin disease. Systemic sclerosis can be diffuse or limited. Limited systemic sclerosis is also called CREST (calcinosis, Raynaud's esophageal dysfunction, sclerodaytyly, telangiectasiae). Scleroderma-like disorders are believed to be related to industrial environment exposure. In Sine disease, there is internal organ involvement without skin changes.
The major manifestations of scleroderma and in particular of systemic sclerosis are inappropriate excessive collagen synthesis and deposition, endothelial dysfunction, spasm, collapse and obliteration by fibrosis.
Scleroderma is a rare disease with a stable incidence of approximately 19 cases per 1 million persons. The cause of scleroderma is unknown. However, the genetic predisposition is important. Abnormalities involve autoimmunity and alteration of endothelial cell and fibroblast function. Indeed, systemic sclerosis is probably the most severe of the auto-immune diseases with 50% mortality within 5 years of diagnosis (Silman, 1991).
In terms of diagnosis, an important clinical parameter is skin thickening proximal to the metacarpophalangeal joints. Raynaud's phenomenon is a frequent, almost universal component of scleroderma. It is diagnosed by color changes of the skin upon cold exposure. Ischemia and skin thickening are symptoms of Raynaud's disease.
Several underlying biological processes are implicated in the initiation, severity and progression of the disease and include vascular dysfunction, endothelial cell activation and damage, leukocyte accumulation, auto-antibody production and crucially, an uncontrolled fibrotic response which may lead to death (Clements and Furst, 1996). Fibroblasts have a pivotal role in the pathogenesis of this disease. Primary fibroblasts obtained from patients with scleroderma exhibit many of the characteristic properties of the disease seen in vivo, notably increased extracellular matrix synthesis and deposition, notably of collagen and fibronectin, and altered growth factor and cytokine production such as of TGFβ and CTGF (Strehlow and Korn, 1998 and LeRoy, 1974).
There is no curative treatment of scleroderma. Innovative but high-risk therapy proposed autologous stem cell transplantation (Martini et al., 1999). In particular, there are currently no treatments for scleroderma targeting the fibrotic process (Wigley and Boling, 2000).
Identification of the genes associated with disease risk and scleroderma progression may lead to the development of effective strategies for intervention at various stages of the disease.
SARP-1 (secreted apoptosis-related protein 1) is a member of a family of secreted proteins known as secreted frizzled related proteins, based on their homology to the cysteine rich domain (CRD domain) found in the frizzled family of 7 transmembrane receptors (Rattner et al., 1997). Frizzled genes were originally identified in drosophila and control tissue polarity (Adler et al., 1987). Frizzled proteins are the receptors for the highly conserved Wnt family of at least 16 secreted signaling molecules that regulate cell-to-cell interactions during embryogenesis (Smalley and Dale, 1999). Insights into the mechanisms of Wnt action have emerged from several systems: genetics in Drosophila and C. elegans; biochemistry in cell culture; and ectopic gene expression in Xenopus embryos. Many Wnt genes in the mouse have been mutated, leading to very specific developmental defects. The Wnt signalling pathway which is triggered by the interaction of Wnt with frizzled proteins, is mediated through several cytoplasmic relay components, and functions to suppress the activity of the multiprotein β-catenin turnover complex, thus allowing a build up of cytosolic β-catenin which then enters the nucleus and forms a complex with TCF to activate transcription of Wnt target genes (Miller et al., 1999; Kühl et al., 2000).
Wnt-frizzled interactions may be modulated through the restricted expression of distinct Wnt binding proteins, the secreted frizzled related proteins (sFRP). SFRPs are able to bind Wnt through the N-terminal CRD domain. They can therefore sequester Wnt away from its receptors and thereby antagonize its effects (Bafico et al., 1999).
SARP-1 is known under several alternative names, such as SDF-5, PRO697, ATG-1622, HLHDY31, SFRP-2. Partial or full length protein and/or nucleic acid sequences of murine or human SARP-1 have been described in several patent applications, e.g. WO 98/35043, WO 98/13493, EP 0 879 887, WO 99/46281.
In terms of function, the secreted frizzled-related proteins (SFRPs) appear to act as soluble modulators of Wnt signaling by competing with membrane-bound frizzled receptors for the binding of secreted Wnt ligands. Apart from SARP-1, the human proteins of this family so far known comprise SARP-2 (SFRP-1) and SARP-3 (SFRP-5). Murine SARP-1, and human SARPs-2 and -3 have been described to have the ability to either sensitize cells to apoptosis or to inhibit the apoptotic response (Melkonyan et al., 1997). When expressed in a breast adenocarcinoma cell line, mouse SARP-1 and human SARP-2 exhibited opposite effects on cell sensitivity to pro-apoptotic stimuli. Whereas cells with SARP-1 had higher resistance, cells expressing SARP-2 were sensitized to apoptosis induced by tumor necrosis factor and ceramide. Expression of SARP-1 or SARP-2 modified the intracellular levels of β-catenin, an indicator of Wnt mediated signal transduction, suggesting that SARPs interfere with the Wnt-frizzled signaling pathway (Melkonyan et al., 1997).
Northern blot analysis revealed that the SARP genes have distinct expression patterns (Leimeister et al., 1998). SARP-1 exists as 2.2- and 1.3-kb transcripts in several human tissues, with the highest levels in colon and small intestine. Chang et al., 1999, reported that SARP-1, or SFRP-2, is highly and preferentially expressed in bovine retina throughout the inner nuclear layer. Within the retina, SARP-3, or SFRP-5, is specifically expressed in the retinal pigment epithelium.
By analysis of somatic cell hybrids, Melkonyan et al. (1997) mapped the SARP-1 gene to human chromosome 4. Chang et al. (1999) refined the map position to 4q31.3 using radiation hybrid analysis.
There is also mounting evidence that an altered SARP-1 expression is related to cancer (WO 98/13493).